Most biological processes involve specific protein-protein interactions. General methodologies to identify interacting proteins or to study these interactions have been developed. The advantages of genetic approaches in drug discovery have recently received increased attention. These advantages include both cost-effectiveness and simplicity.
Among these methods, the yeast two hybrid system currently represents the most powerful in vivo approach to screen for polypeptides that could bind to a given target protein. It is also equally suitable for the detection of both homo- and heterodimeric protein interactions. These technologies have originally been developed by Fields et al. (Fields and Song, 1985, Nature, 340, p. 245-246, “A novel genetic system to detect protein-protein interaction”).
The yeast two-hybrid system utilizes hybrid proteins to detect protein-protein interactions by means of direct activation of a reporter-gene expression. In essence, the two putative protein partners are genetically fused to the DNA-binding domain of a transcription factor and to a transcriptional activation domain, respectively. A productive interaction between the two proteins of interest will bring the transcriptional activation domain of an adjacent reporter gene (usually LacZ or a nutritional marker) giving a screenable phenotype. The transcription can be activated through the use of two functional domains of a transcription factor: a domain that recognizes and binds to a specific site on the DNA and a domain that is necessary for activation (Keegan et al., 1986, Science, 231(4739): 699-704 Separation of DNA binding from the transcription activating function of eukaryotic regulatory protein).
To date however, the two-hybrid assay system has not been specifically applied to the systematic study of prokaryotic protein-protein interactions although number of diseases are due to prokaryotic microorganisms.
One of the prokaryotic microorganisms presenting a great interest is Helicobacter pylori. Helicobacter pylori (H. pylori) is a microaerophilic, Gram negative, slow growing, spiral shaped and flagellated organism. H. pylori has been first isolated in 1983 from gastric biopsy specimen of patient with chronic gastritis (Marshall et al., 1984, Lancet, i:1311-1314, Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration).
Helicobacter pylori has become identified as a primary cause of chronic gastroduodenal disorders, such as gastritis, dyspepsia, and peptic ulcers, in humans. Studies have shown (Labigne et al.) that H. pylori can be successfully eradicated by a treatment combining two antibiotics with a proton pump inhibitor. However, few antibiotics are active against H. pylori, and antibiotic-resistant strains have begun to appear.
H. pylori strain n° 26695 genome has been studied by Tomb et al. (Tomb et al., 1997, Nature, vol. 388, 539-547, The complete genome sequence of the gastric pathogen Helicobacter pylori). This strain's genome consists of a circular chromosome with a size of 1,667,867 bp, average G+C content of 39%, and 1590 predicted coding sequences (open reading frames or “ORF”).
The bacterial factors necessary for colonization of the gastric environment, and for virulence of this pathogen, are poorly understood. Examples of known virulence factors are:                Enzymes involved in neutralizing the acid gastric pH: the multisubunit urease is a characteristic enzyme that is crucial for survival in acidic pH and for successful colonization of the gastric environment, a site that few other microbes can colonize (Labigne et al., WO 93/07273, Helicobacter pylori genes necessary for the regulation and maturation of urease, and use thereof). Genes encoding ureases have been located on a 34 kb chromosome fragment and comprise ureA, ureB, ureC, ureD, ureE, ureF, ureG, ureH and ureI.        Bacterial flagellar proteins responsible for motility across the mucous layer (Hazell et al., 1986, J. Inf. Dis., 153, 658-663 Campylobacter pyloridis and gatritis: association with intracellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium; Leying et al., 1992, Mol. Microbiol., 6, 2863-2874 Cloning and genetic characterization of Helicobacter pylori flagellin gene): flagellar filaments biosynthesis comprises A and B flagellins and the filament cap. These two biosyntheses are regulated by flbA gene (Suerbaum et al., French patent application, 1995, N 2 736 360, Cloning and characterization of flbA gene of Helicobacter pylori, aflagellated strains production).        Two other essential toxins for virulence are VacA and CagA:                    VacA is a H. pylori toxin that induces the formation of large acidic vacuoles in host epithelial cells. These large vacuoles originate from massive swelling of membranous compartments of late stages of the endocytic pathway (de Bernard et al., 1997, Microbiology, 26(4), 665-674, Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol). Proof for receptor-mediated interaction with VacA has been made by Pagliaccia et al.; m2 allele of vacA gene has always been described as inactive in the in vitro HeLa cell assay, however, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence (Pagliaccia et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95(17), 10212-10217, The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity).            CagA is one of the proteins encoded by the “cag pathogenicity island” (Spohn et al. 1997, Molecular Microbiology, 26(2), 361-372, Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori) found in H. pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma. CagA is produced by 50-60% of H. pylori strains; it is a high molecular weight (120-140 kDa) superficial protein and an immunodominant antigen with unknown function. H. pylori strains that produce CagA protein have two genes cagB and cagC (36 and 101 kDa proteins, respectively). These genes are highly associated with duodenal ulcers (Blaser et al. 1996, WO 96/12825, cagb and cagC genes of Helicobacter pylori and related methods and compositions).                        Other virulence factors are: several gastric tissue-specific adhesins (Boren et al., 1993, Science, 262, 1892-1895).        
Therapeutic agents are currently available that eradicate H. pylori infections in vitro. However, methods employing antibiotic agents result in the emergence of bacterial strains which are resistant to these agents.
As number of diseases are due to prokaryotic microorganisms, there is a great need for new tools directed to the functional and global study of these newly characterized complete or partial genome, particurlarly Escherichia coli genome, but also of pathogenic microorganisms such as H. pylori, Staphylococcus aureus and Streptococcus pneumoniae genomes.
In addition to the need for these new tools, there is also and especially a need to find new E. coli, H. pylori, S. aureus and S. pneumoniae protein-protein interactions for the development of more effective and better targeted therapeutic.